A stable molecular antioxidant activity was observed, since 100-µM MeO-MBM was measured to be 11.2% in the DPPH assay, with a decrease to 9.7% after irradiation. In comparison, the molecular antioxidant activity of 100-µM avobenzone was determined to be 0.8%. In keratinocytes, MeO-MBM reduces the intracellular ROS by 90% and avobenzone by 75% with tBHP as the inducer and by 53% and 57%, respectively, when induced by pyocyanin, indicating the redox scavenging capacity of both these molecules. These results indicate that MeO-MBM functions initially as an antioxidant material and as a photoantioxidant during its conversion process to avobenzone. This research provides insight into the development of active ingredients for topical applications with dynamic functionalities. Using this approach, we demonstrate the possibility to extend the UV protection offered to skin cells while combating cellular stress in parallel.Avobenzone, one of the most commonly used UV filters in topical sunscreens, is susceptible to photodegradation with a consequential reduction of its UV absorbing properties. This loss of function may lead to skin irritation, photodermatosis, and photoallergic reactions caused by photodegradation byproducts. In this work, we aim to address this issue with a substance named methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, but which converts to avobenzone, a known and approved UVA filter, under mainly UVB light irradiation. The antioxidant and intracellular radical formation properties of MeO-MBM were compared to the ones of avobenzone. The UV irradiation of MeO-MBM led to an increase in UV absorption primarily in the UVA range after conversion, both in vitro and in vivo. HPTLC and UHPLC studies illustrate the conversion of MeO-MBM to avobenzone in vitro after irradiation at 250 kJ/m Avobenzone  is the most commonly used UVA filter in sunscreens, but it degrades irreversibly when exposed to sunlight or other UV light sources ,3,4,5,6.The irreversible UV-driven photodegradation is based, in part, on the photoexcitation of avobenzone, leading to a nonadiabatic population of high-lying S0 vibrational levels and, subsequently, to isomerization and, thus, to a less photostable form . PhotolyTo limit the stress and damage caused by UV irradiation to skin tissue, sunscreens with filter materials, such as avobenzone, are used that absorb energy in the form of electromagnetic radiation. It is also possible to add a substance to aid the filter material in sunscreen formulation. Such a booster material in sunscreens is defined as an enhancer of UV-ray protection, maintaining or, ideally, reducing the quantity of the filter material needed while increasing the filter’s shielding performance, with the aim of increasing the sun protection factor (SPF) . ChemicaThus, there is a need for chemical auxiliary materials that stabilize UV filters and supports their function, as well as prevent cellular stress caused by degradation byproducts and the environment. Furthermore, it would be ideal if one substance could address all these issues simultaneously, thereby having multidimensional functionality. The following study will show that MeO-MBM is able to fulfill all three required functionalities and is a suitable active ingredient in sun care formulations, with the capability to convert into an approved UVA filter.The aim of using MeO-MBM is not to substitute avobenzone. A UV filter requires registration to assure the efficacy of the sun filter. MeO-MBM has no UV filter properties as such and, thus, is not suitable for registration. In the case of avobenzone in the final formulations, there is a regulatory concentration limit. Therefore, the intention is to add an additive that either stabilizes avobenzone against the already known photodegradation, boosts the effect of avobenzone, or use a material that converts during UV exposure to avobenzone, like the here-described MeO-MBM.MeO-MBM releases two hydrogen radicals that stoichiometrically compensate and neutralize the formed radicals 4-methoxybenzaldehyde and 1-(4-tert-butylphenyl) ethenone from the Norrish-type I degradation of avobenzone. This molecule also possesses radical scavenging moieties that limit the degradation of avobenzone, as well as reduce cellular oxidative stress. Further research into such active ingredients with multidimensional functionality would help simplify the topical formulation, reduce the adverse effects, and possibly lead to a higher adherence to the application of sunscreens, leading to the prevention of skin diseases. A deeper understanding of the molecules that can be combined and the biochemical processes that they can support will also need to be undertaken.The aim of a transformer material is to slow down the UV-dependent diminution of the SPF, ideally directly on human skin. This is achieved by the conversion of MeO-MBM, which is not a UV filter material, into avobenzone via UVA/B exposure. After further UVA/B irradiation, the photoketonization process of avobenzone is initiated, leading to the Norrish-type I cleavage . This phMeO-MBM protects avobenzone from degradation as the 4-methoxybenzaldehyde radical and 1-(4-tert-butylphenyl) ethenone radical may react with oxygen, presumably causing an accelerated degradation of avobenzone. The two hydrogen radicals raised from MeO-MBM photolysis under UV light play an important role in stabilizing avobenzone by neutralizing of formed radicals. With the potential of MeO-MBM to convert into avobenzone and maintain the concentration of an active sun filter on human skin, thus maintaining the SPF, MeO-MBM compensates for the UV irradiation-induced degradation of the material without functioning itself as a UV filter. In the following experiment, the conversion of MeO-MBM is tested in vitro.Before the irradiation, MeO-MBM did not absorb UV light above an intensity of 0.05 AU  coupled with a charged aerosol detector (CAD) showed a single peak at tt = 8.22 a. A sampt = 8.22 b. After t = 8.22 c. In theThese findings strongly support the hypothesis that MeO-MBM is converted into avobenzone after irradiation. This conversion would enable the protection of the skin from DNA damage, UV-induced skin changes, or extrinsically induced aging. In the next investigation, this conversion was tested in vivo on human skin.The UV absorption in vivo was measured utilizing remission spectroscopy. When light penetrates the skin’s surface, radiation is scattered in the various layers of the skin like the epidermis or dermis, and the diffuse reflection is measured as remission. Based on the determination of the changes in the remission spectroscopy before and after UV irradiation, the efficacy was evaluated concerning its absorption properties. The remission measurements were analyzed within the spectral range between 280 and 400 nm.The tested formulation with and without MeO-MBM (placebo) was a formulation containing a mixture of UVB filters that provided SPF 10 in vivo. Untreated skin marginally absorbed UV light between 280 and 400 nm . The plaAfter demonstrating the conversion properties of MeO-MBM after UV irradiation in vitro and in vivo, the antioxidant properties and intracellular radical formation of MeO-MBM were compared with avobenzone to underline its properties in protecting living skin cells against UV-induced damage.Cellular stress in skin is caused by multiple stressors like UV irradiation, allergens, temperature, sweat, nutrients, pollutants, and age. Cellular processes and organelles like mitochondria are negatively affected by the accumulation of ROS and RNS. To improve the cellular health in human skin, antioxidative ingredients are preventively used to avoid radical-induced molecular and cellular damage. In this context, the antioxidative potential of MeO-MBM was evaluated based on the ability to inhibit 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) in vitro compared to the antioxidative potential of avobenzone and irradiated MeO-MBM. In addition, we tested the formation of ROS and RNS at the cellular level, which are produced during UV-induced stress.50 values of 11.8 µM and 4.4 µM, respectively, the observed antioxidant effect appeared to be less pronounced [In the DPPH assay, the antioxidative effect of MeO-MBM at 10 µM was 2.4% and, at 50 µm, increased threefold to 7.4%, further increasing at 100 µM to 11.2% . For irronounced .Based on the reaction shown in p-values. For the formation of RNS, a significant difference was observed, as MeO-MBM was 41% more potent in reducing l-arginine-induced radicals in HaCat cells (l-arginine to l-citrulline by nitric oxide synthases (NOS) and nonradicals like peroxynitrite (ONOO−). RNS causes cell damage through structural alterations of proteins and DNA, consequently inhibiting the enzymatic activity and interfering with the regulatory functions of the macromolecules [These experiments were conducted to compare the intracellular ROS and RNS scavenging activity of MeO-MBM and avobenzone. Considering the PBS control  as no reat cells C. The RNolecules ,26,27. Molecules . Peroxynolecules . The dat20H24O3 and a molecular weight of 312.41 g/mol with the structure (MeO-MBM—3-(4-tert-butylphenyl)-3-hydroxy-1-(4-methoxyphenyl) propan-1-one (CAS Nr. 955359-34-9), with the molecular formula Ctructure , was syn® 80) were obtained from Merck KGaA . Sandblasted polymethylmethacrylate (PMMA) plates were purchased from Schoenberg GmbH & Co KG . All solvents were of analytical grade obtained from Merck KGaA . The working solutions were prepared immediately prior to the measurements.DPPH was purchased from Sigma-Aldrich . Avobenzone, isopropylmyristate, and Polyoxyethylen(20)-sorbitan-monooleat (Tweenw/w) and applied on 19-cm2 PMMA plates at a concentration of 1 µL/cm2. The conversion of MeO-MBM into avobenzone in vitro was carried out in a suntest device  with an effective UV intensity of 36 W/m2 and a UVB/UVA ratio = 1:20. The tested UV doses were 250 kJ/m2 (116-min irradiation time) and 500 kJ/m2 (232-min irradiation time), respectively. Active cooling of the plates from below and air cooling from above ensured that the temperature never exceeded 25 °C for the entire measurement period. After irradiation, the samples were extracted with 50-mL isopropanol from the PMMA plates. The absorbance was measured between 290 and 400 nm using a UV–Vis spectrophotometer  in a 1-cm cuvette.MeO-MBM was dissolved in isopropyl myristate  were dissolved in ortho-xylene and applied on a 19-cm2 PMMA plate at a concentration of 1 µL/cm2. Irradiation was carried out in a suntest device  at 250 kJ/m2, followed by extraction of the substances from the plates with 2-mL ethanol before analyzing. Equally treated samples were additionally stored in the dark without irradiation as a comparison.MeO-MBM and avobenzone  with an automatic sampler  as bands with 6.0-mm widths. The plates were developed in toluene:methanol (9:1 ratio). The chromatograms were photographed at the 254-nm wavelength . The retardation factor (Rf) value was obtained by using the software winCATS 1.4.8 .HPTLC analyses were carried out by applying the sample solutions onto high-performance thin-layer chromatography (HPTLC) plates  and gradient elution with solvents A: water/formic acid 99.9/0.1 (v/v) and B: acetonitrile/formic acid 99.9/0.1 (v/v). The gradient started at 45% (v/v) B, ramping up to 70% over 10 min, then ramping down to 45% over 2 min and being held for 3 min. CAD detection was performed at a 35 °C evaporation temperature, 30 °C column temperature, 0.8-mL/min flow speed, and 5-µL injection volume. The conversion of MeO-MBM to avobenzone was calculated based on their concentration (ß) present in the irradiated sample. The concentration was calculated from a five data point calibration curve of pure MeO-MBM and avobenzone, respectively (0–150 µg/mL). The conversion was calculated using Equation (1).The samples used for analysis by HPTLC were also used for the identification and quantification of MeO-MBM and avobenzone by UHPLC  coupled with a CAD . Separation was accomplished using a C-18 column (Chromolithw/w) was incorporated additionally, while the formulation without MeO-MBM served as the placebo. The formulations were applied on the back of ten volunteers . Remission measurements were carried in the rage of 240–400 nm. Stimulating radiation intersected the skin at a 90° angle. On the skin, the light beam had a diameter of approx. 12–14 mm. Remitted radiation was recorded under a 45° angle. This 90°/45° geometry was used to suppress the reflected light radiation that was detected together with the remission signal during remission spectroscopy. Skin remission spectra were first adjusted using a barium sulphate spectrum that served as a standard of 100% reflectance. The remission spectroscopy measurements were performed prior to the product application. A solar simulator  was used as a light source. The excitation spectrum of the light source complied with the international SPF Test method (COLIPA spectra). The measurements were performed on each test area 15 min after product application (t1) and UV irradiation (t2) with 8 times the minimal erythema doses (8 MED), resulting in an actual 0.8 MED due to the SPF 10 formulation. The changes in remission were determined as the difference between before and after irradiation. Absorption spectra were calculated from the differences in remission.The topical Oil-in-Water (O/W) test formulation contained a mixture of UVB filters  to provide SPF 10 in vivo. MeO-MBM  were mixed with a DPPH solution , resulting in a 10-, 50-, or 100-µM substance sample (S) reacting with 0.10-mM DPPH. After a 60-min reaction time, the reduction of the DPPH radicals was measured by a decrease in the absorption (A) at 515 nm using a UV–Vis spectrophotometer . An ethanolic 0.1-mM DPPH solution was used as the control sample (CS). All assays were performed in triplicate, and the results were expressed as the % inhibition of 0.1-mM DPPH. The inhibition was calculated following Equation (2).® 80 was selected as the negative control and set to 0% viability. When treated with the test substances MeO-MBM and avobenzone at a concentration of 100 µM, HaCat cells did not show a decrease in viability greater than 20% compared to the control. Hence, this concentration was used in further biological testing.First, the cytotoxicity of the test substances was evaluated, and the influence on cell viability was performed as recommended in the user manual using the ATPlite 1step luminescence assay . Cell culture media was used as a positive control set to 100% viability. A 7.5% Tween5 cells/mL and incubated at 37 °C and 5% CO2. After 24 h, the cell culture medium was removed, and the cells were washed with 200-µL washing buffer, followed by treatment with the test substances, or 5-mM N-acetylcysteine as a negative control was added for the next 24 h. After which, 50 µL of the inducer were added to the wells and incubated at the same conditions for 24 h. The inducer concentrations were as follows: 1-mM Pyocyanin, 40-mM tBHP, and 100-µM l-arginine. one hundred microliters of ROS/superoxide detection solution were added to each well. The plate was measured using a fluorescence microplate reader  with an excitation at 488 nm and an emission at 520 nm.The ROS were analyzed using the ROS-ID Total ROS/Superoxide Detection Kit , as per the manufacturer’s instructions. Briefly, HaCat cells were seeded in a 96-well plate at a density of 1.25 × 10In this study, we showed that MeO-MBM has multifunctional properties as an active ingredient to protect skin against reactive radical formation. It has been shown that MeO-MBM converts into avobenzone in vitro and in vivo after having been exposed to photons. MeO-MBM per definition releases based on the structural changes during the conversion to avobenzone two hydrogen radicals. The hypothesis is that the release of the two hydrogen radicals stoichiometrically directly compensate and neutralize the formed radicals 4-methoxybenzaldehyde and 1-(4-tert-butylphenyl) ethenone from the Norrish-type I degradation of avobenzone or further react with the radical species present in human UV-stressed skin, and this is referred to as photoantioxidative functionality. MeO-MBM exhibited antioxidant activity in the DPPH test, while, in cellular irradiation  during t


Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid  Since  of carbon ions (12C6+). The carbon ion energy was measured as 80 MeV/u, and the average linear energy transfer (LET) value was 31 keV μm−1. After irradiation, the cells were placed on seed medium plates to assess the mortality. Cell survival rate = colony counts of irradiated cells/colony counts of irradiated cells with 0 Gy × 100%. And cell mortality = 100% – cell survival rate.Cells were inoculated into the seed medium where the ALE process commenced. Then the temperature of the medium was gradually decreased from 16 to 4°C with 4°C per change . When thBefore the second-round ALE, the cell mortality was firstly assessed with different concentrations  of quizalofop-p-ethyl in the agar plates containing seed medium. Cell survival rate = colony counts with different doses of quizalofop-p-ethyl/colony counts with 0 μM quizalofop-p-ethyl × 100%. And cell mortality = 100% – cell survival rate. Then the ACCase inhibitor quizalofop-p-ethyl was used for the second-round ALE. The concentration of quizalofop-p-ethyl was gradually increased from 20 to 100 μM with 20 μM per change. After the growth rate reached the maximum at 100 μM quizalofop-p-ethyl, the evolution proceeded for 10 cycles for ~60 days. Then the endpoint strains were plated on seed medium plates to select the single colonies.Aurantiochytrium were harvested and immersed in RNAlock Reagent , and stored at −80°C until use. Total RNA was isolated using TRIzol reagent  according to the manufacturer's instructions. RNA purity was checked using a Nano-300 spectrophotometer , and RNA degradation and contamination was monitored on 1% agarose gels. Synthesis of cDNA was carried out with a Revert Aid First strand cDNA Synthesis Kit (Thermo Scientific), and cDNA was used as the template for qRT-PCR analysis with the primers listed in Briefly, cells of Biomass was expressed as dry cell weight (DCW). Five milliliters samples were harvested and freeze-dried to constant weight at −50°C. The glucose concentration was analyzed with a SBA-40E Biosensor .Total lipid was extracted using a combination solvent of chloroform and methanol . The extracted lipids were transferred to a pre-weighed glass tube and vacuum evaporated under 50°C, then the total lipid was weighed. To obtain the fatty acid methyl esters (FAMEs), the total lipid was dissolved in 1 mL of chloroform and incubated with 2% (v/v) sulfuric acid/methanol at 85°C for 2.5 h. FAMEs were determined by gas chromatography  .Kjeldahl nitrogen determination method was employed in determining the protein content . LyophilThe carbohydrate content was assayed following the Phenol-Sulfuric acid method. Lyophilized samples were ground using a mortar and then hydrolyzed in boiling water with 6 M hydrochloric acid for 0.5 h. After cooling, 10% (w/w) NaOH was used to regulate pH = 7.0. Hydrolysate was then taken for carbohydrate content determination by the Phenol-Sulfuric acid method .2PO4, 1 g/L MgSO4, and 15 g/L artificial seawater. One milliliter of antifoam, THI®X-298 , was added at the beginning to control foam formation. Samples (50 mL) for off-line determination of biomass, glucose, lipid, and fatty acid profiles were drawn at 12 h intervals until the end of the fermentation.The fed-batch fermentation experiment was performed in a 5 L Biostat® B plus bioreactor as described previously . The culp < 0.05 and p < 0.01) was assessed using a t-test.All data are the means of three replicates and reported as the mean ± SE. The statistical analysis was carried out by Excel, and the significance of differences , a dose-dependent mortality was observed  is capable of catalyzing the first bottleneck step in fatty acid biosynthesis. As reported in previous studies, the lipid production in oleaginous microorganisms could be increased by the enhanced activity of ACCase , 39. Qui ~2-fold . Another ~2-fold . Therefo96 single colonies were taken and then cultured in fermentation medium for 96 h. Lipid profiles of these strains were screened  to selecAurantiochytrium sp. SD116 to enhance the DHA purity and yield  heaAs shown in Aurantiochytrium sp. is characterized by two distinct physiological stages, namely the growth phase and the oleaginous phase. Therefore, the transcription profiles at 48 and 72 h which represented these two phases, respectively, were monitored. During the growth phase, the oleaginous microorganisms convert the carbon source into cell mass, which is rich in proteins, but poor in quantities of lipids , isocitrate dehydrogenase (ICDH), and malic enzyme (ME) were significantly increased in E-81 strain than the other. CS and ICDH are the key enzymes in TCA cycle, whereas ME is the key enzyme in pyruvate malate shuttle. Both of these pathways are known to generate biosynthetic precursors which are involved in the production of energy or reducing power that are essential for the biosynthesis of various macromolecules. Therefore, the increased transcription levels of CS, ICDH, and ME in E-81 strain may provide more biosynthetic precursors.The growth cycle of f lipids . As showand OrfC  were notCS and ME were up-regulated while no changes of the transcription level of ICDH was observed in E-81 strain, suggesting that more acetyl-CoA could be synthesized in E-81 strain than SD116. The accumulated pool of acetyl-CoA along with reducing power could increase lipid and DHA productivities , which could produce UDP-glucose as a substrate to synthesize carbohydrate, was detected here , National Natural Science Foundation of China , the Shandong Province Natural Science Foundation (ZR2020QD099), the Key Deployment Project of Centre for Ocean Mega-Research of Science, Chinese Academy of Sciences (COMS2019J07), Qingdao independent innovation major project (Grant No. 21-1-2-23-hz), and supported by QIBEBT (Grant: QIBEBT I201933). This study was also supported by Dalian National Laboratory for Clean Energy (DNL), CAS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic with significant mortality. Accurate information on the specific circumstances of death and whether patients died from or with SARS-CoV-2 is scarce. To distinguish COVID-19 from non-COVID-19 deaths, we performed a systematic review of 735 SARS-CoV-2-associated deaths in Hamburg, Germany, from March to December 2020, using conventional autopsy, ultrasound-guided minimally invasive autopsy, postmortem computed tomography and medical records. Statistical analyses including multiple logistic regression were used to compare both cohorts. 84.1% (n = 618) were classified as COVID-19 deaths, 6.4% (n = 47) as non-COVID-19 deaths, 9.5% (n = 70) remained unclear. Median age of COVID-19 deaths was 83.0 years, 54.4% were male. In the autopsy group (n = 283), the majority died of pneumonia and/or diffuse alveolar damage . Thromboses were found in 39.2% (n = 62/158 cases), pulmonary embolism in 22.1% (n = 56/253 cases). In 2020, annual mortality in Hamburg was about 5.5% higher than in the previous 20 years, of which 3.4% (n = 618) represented COVID-19 deaths. Our study highlights the need for mortality surveillance and postmortem examinations. The vast majority of individuals who died directly from SARS-CoV-2 infection were of advanced age and had multiple comorbidities. As of May 18th, 2021, the outbreak of SARS-CoV-2 has spread to all continents, with about 164 million confirmed cases and over 3.4 million fatalities worldwide after contracting the respiratory virus4.Severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was first identified in December 2019 in Wuhan, Hubei Province, ChinaInternationally, the COVID-19 pandemic demonstrates that recording of total mortality in a dynamic infection event represents a particular challenge for harmonization and comparability of infection and case fatality figures. For this purpose, a systematic SARS-CoV-2 mortality monitoring has been established at the Institute of Legal Medicine (ILM) of the University Medical Center Hamburg-Eppendorf, Germany (UKE) in March 2020. At that time, there was little knowledge about the causes of death in such fatalities and the question was raised whether the patients died from or with SARS-CoV-2.8. Therefore, systematic investigations of SARS-CoV-2 associated deaths, defined as fatalities associated with a positive SARS-CoV-2 PCR test, deemed necessary to provide evidence for epidemiological clusters and patient cohorts underlying particular hazards for fatal courses of the disease.Considering the possible effects of SARS-CoV-2 on various organs, detailed knowledge of the organotropism of the virus, the identification of risk factors and the underlying ultimate causes of death were of particularly high clinical relevanceThe aim of this systematic postmortem evaluation was to classify all known SARS-CoV-2 associated deaths in the city of Hamburg, Germany, as COVID-19 or as non-COVID-19 deaths and to compare both groups, with respect to demographic, anthropometric and medical characteristics.A total of 735 SARS-CoV-2 associated deaths were analyzed. Conventional autopsies were performed in 38.5% (n = 283). 5.6% (n = 41) of deaths were investigated by usMIA. In total, 55.9% (n = 411) received a PMCT and 35.9% (n = 264) were classified by medical record review only. In 34.6% (n = 254) several examinations were performed consecutively. CA was able to assign a definite cause of death in 99.3% of cases (281/283), followed by usMIA at 90.2% (37/41), isolated PMCT at about 87.9% (138/157) and medical record alone at about 83.0% (219/264)  were classified as COVID-19-related, including 254 (41.1%) by CA. The remaining 15.9% (n = 117) were divided into non-COVID-19 deaths and unclear causes of death . Unclear cause of death was the most frequent (64.3%) in cases limited to medical record review only due to insufficient data and/or missing consent to postmortem examination.Overall , 6.2% (n = 41) of patients died at home and 24.4% (n = 162) in nursing homes. The majority of 68.4% (n = 445) died in hospital, of which 38.9% (n = 259) died on the normal ward, 29.2% (n = 194) in the intensive care unit and 0.3% (n = 2) in the emergency room. In 7 cases, there was no or unclear information. The overall proportion of nursing home residents of all deaths (N = 735) investigated was 52.9% (n = 389). Regarding the survival time of the whole collective, we found a substantially longer time interval of first positively confirmed swab PCR test until date of death of hospitalized patients from May 2020 on , but no comparable increase in outpatients . Tables 54.4% (n = 336) of the COVID-19 death group were men. The median age was 83.0 years (IQR 76.0–88.0) with a higher age of women compared to men (median 84.0 [IQR 78.0–89.0] versus 81 [IQR 74.0–87.0] years), see Table 28.5% (n = 176) died in the outpatient and 70.6% (n = 436) in the hospital setting, with more patients died on normal ward  than in the ICU .82.8% (n = 322) nursing home residents were defined as COVID-19 deaths representing 52.1% of all COVID-19 deaths (n = 618) in our cohort  were defined as non-COVID-19 deaths  and other organ failures led to death in the autopsy group. In 158 (62.2%) COVID-19 cases and in 11 (40.7%) non-COVID-19 cases detailed preparation of the lower extremities took place. In this subcohort, thromboses were found in 39.2% (n = 62) of 158 cases and likewise pulmonary embolisms were found in 22.1% (n = 56) of 253 cases  Table . Among t2 (IQR 20.4–28.8). In 50 CA cases BMI calculation was deferred because of severe edema or body weight or height was missing. 21.1% (n = 43) of the deceased were obese with no difference compared to non-COVID-19 deaths (p = 0.194), see Table The median BMI evaluated in 204 cases was 24.7 kg/mSupplemental Table 9.In contrast to the ILM reported death cases by the date of death, the death cases reported by the Robert-Koch-Institute (RKI), the German government’s central scientific institution in the field of biomedicine, show a higher peak mostly at the beginning of the weeks depending on the official reports Fig. . Overall11 this corresponds to an increase in deaths of about 5.5% for 2020 alone, of which 3.4% (n = 618) represent COVID-19 deaths 15. Epidemiologically, men have a higher risk of severe COVID-19 sequelae than women16. The number of confirmed positive cases is roughly equal in men and women17, but the male-dominated sex ratio in COVID-19 deaths has been confirmed in our study and worldwide19.Our main results show that the majority of deaths associated with SARS-CoV-2 positivity died due to COVID-19 (84.1%), which is consistent with previous reports20 described fewer comorbidities, an average of 1.6 for the German population, figures that were related to reported deaths by the RKI.The deceased in our autopsy group suffered from many pre-existing conditions, especially of cardiovascular origin. The autopsy group of COVID-19 deaths had an average of 2.9 comorbidities. In contrast, Rommel et al.23. Here, our results show a heterogeneous distribution between BMI values. Interestingly, only 21% of the COVID-19 death group were obese. This roughly corresponds to the national average for Germany, as about 22% of the German population had a BMI ≥ 30 in 2017 (most recent data)24. It should be noted that older patients in general, especially in our cohort of nursing home residents, are more prone to cachexia, malnutrition or underweight, which may also be an underestimated risk factor.However, these data were evaluated purely anamnestically; a post-mortem examination was not carried out. Previous studies have also reported that obesity is a relevant pre-existing condition20. Although women in the COVID-19 death group were older than men, there were no differences in sex, age or type of comorbidity when comparing the COVID-19 to the non-COVID-19 death group, highlighting the influence of underlying risk factors for fatal disease outcome in older patients. Notably, pre-existing neurological conditions were common in the hospitalized COVID-19 death group.Our data confirm the above studies that age, gender and comorbidities are risk factors for fatal outcome26. The virus infects airway epithelial cells27, leading to diffuse alveolar damage, edema and a marked increase in lung weight in our COVID-19 autopsy group, as previously reported28. It is noteworthy that the histological changes in the lungs were heterogeneously distributed, corresponding with our radiological findings of patchy dullness opacities, which most likely indicate a diffuse spread of the virus in the respiratory tract29. In addition, multi-site organ tropism has been reported to be favored in tissues with high expression of the angiotensin converting enzyme 2 receptor36. As venous thromboembolism was increasingly seen as a complication, since the beginning of May 2020, adapted anticoagulation has also been used in intensive care in Hamburg28. Although pulmonary emboli and deep vein thrombosis of the lower extremities were still diagnosed in some of the autopsies. The survival time of hospitalized patients increased from May 2020 onwards.The main cause of death in our cohort and others was SARS-CoV-2 induced lung injury37. 28.5% of COVID-19 deaths died in the outpatient setting, which underlines the need for close cooperation between the disciplines involved. The RKI figures show a comparable distribution of places of death as in our cohort, with about 25% of deaths occurring outside the hospital38. It was notable that many cases in the non-COVID-19 death group occurred in the outpatient setting, mostly due to minor or non-specific symptoms of disease.Notwithstanding the important role of pathology in clarifying the cause of death in clinical cases, our results show the high importance of forensic autopsy in the context of pandemics39.Interestingly, Hamburg had a slightly increased excess mortality rate of about 4.4% compared to 2016–2019 and 5.5% compared to the last two decades in 202020. About 3/4 of these higher numbers can be explained by the number of COVID-19 deaths in 202020. Other indirect effects such as threshold increases for the utilization of outpatient treatment or hospitalizations due to other diseases may have additionally influenced the total number.Compared to Hamburg, excess mortality was also found nationwide, consistent with the number of people who died from or with SARS-CoV-2 by the end of September 202041. The need for a standardized, nationwide recording method is illustrated by a difference of 14 cases between RKI and ILM of Hamburg deaths by the end of 2020 (632 vs. 618 cases)10.Reliable information on mortality is therefore of paramount importance to establish sound public health policies and to literally fight the pathogens of emerging infections. Previous and ongoing pandemics have shown that autopsy is a powerful tool to understand the underlying pathology of a disease46. Compared to this percentage, the autopsy rate of 38.5% reported in this study appears exceptionally high. Interestingly, the autopsy cohort did not have any variables that were statistically different from the overall cohort. It is therefore conceivable that due to the high autopsy rate in our study, the findings made for CA, e.g., on comorbidities and BMI, are transferable to the overall cohort of COVID-19 deaths.Accurate postmortem diagnosis during the initial phase of an emerging epidemic represents an improvement in the identification of the specific etiological agent, which has significant implications for disease surveillance. Although our data show that CA is the most accurate way to assess the cause and manner of death, autopsy rates worldwide have declined significantly in recent decades and are below 5% in Germany49. In contrast, usMIA is flexible, less expensive and has also been tested with promising results and own experience55. This methodology represents a research method that can be useful, especially in countries where mortality data are not available, to counteract the loss of numbers from CA in one's own setting57. Notwithstanding the advantages of minimally invasive autopsy, there are diagnostic limitations of this approach, particularly due to the accuracy in localizing pathological findings to ensure representative sampling. Further studies are needed to demonstrate concordance between CA and MIA and thus to verify the reliability of MIA.To further increase the number of morphology-based postmortem examinations, usMIA was implemented as an alternative to CA. Other imaging modalities, including CT, MRI and/or robotic biopsy collection, can only be performed in centers of excellence and require significant budgets and infrastructureFirstly, only part of the cohort was subjected to CA. A relevant part of the assessment was based only on the evaluation of medical records and available documents. Secondly, a shorter PMI (72 h) was assumed as a prerequisite for performing a CA or usMIA  during follow-up for organ samples and further laboratory approaches. Unfortunately, numerous cases did not fulfil this qualitative preselection because they were reported too late to the ILM or were not known. Thirdly, the diagnostic efficiency of a combined usMIA/PMCT as an evaluation method needs to be compared with CA in further studies. Finally, slight differences in individual assessment may have occurred due to interindividual perception and subjective interpretation of the morphological findings.Accurate information on the specific causes of death in patients dying from emerging infections in particular is scarce, which is why the question of whether patients died from or with SARS-CoV-2 has arisen since the start of the pandemic in 2020.We show here that the vast majority died directly from SARS-CoV-2 infection as COVID-19 deaths, had advanced age and multiple comorbidities.The multimodal "Hamburg Way", i.e., a systematic evaluation of all SARS-CoV-2-associated deaths in the city of Hamburg, only became possible through close cooperation with the Department of Social Affairs and Health, which led to an adjustment of the medical treatment of COVID-19 patients in the early phase of the pandemic. In addition, the systematic recording of non-clinical deaths led to a comprehensive population-based recording and evaluation of deaths. However, such an evaluation would not have been possible without the consent of the relatives to the scientific evaluation and the direct networking with the local health offices and special legal foundations in Hamburg.All reported SARS-CoV-2-associated deaths (defined as ante- and/or post-mortem confirmed SARS-CoV-2 infection of a person at any point in time) of Hamburg citizens were evaluated at ILM Hamburg in collaboration with the health authorities in the period from mid-March 2020 to 30th September (first wave) and 1st October to 31st December 2020 (second wave).58. Figure To scrutinize the reports and in order not to overlook any unknown cases, all deceased admitted to ILM were screened for viral SARS-CoV-2 RNA using a throat swab followed by immediate RT-qPCR at the Institute of Microbiology, Virology and Hygiene, UKE as previously describedDemographic , and medical characteristics (cause of death) were collected for the overall collective. Further anthropometric (BMI) and medical/autopsy characteristics  were added for the autopsy sub-group.Institutional review board approval from the independent ethics committee of the Hamburg Chamber of Physicians was obtained for this study (reference numbers 2020-10353-BO-ff and PV7311). The study complied with the tenets of the Declaration of Helsinki. Informed consent was obtained from a next of kin or legal representatives and authorities for the death case evaluation. All data were pseudonymized according to the guidelines from the central ethics commission of the federal medical association.Depending on the order and the consent of the relatives, a four-step concept was established to determine the underlying cause of death and thus categorize the individual cases into COVID-19 and non-COVID-19 deaths. The final categorization of each case was done by consensus with a supervisor's decision.conventional autopsy (CA) through opening of all three body cavities.ultrasound-guided minimally invasive autopsy through ultrasound-guided needle puncture of the organs .48.postmortem computed tomography (PMCT) of the whole body  in accordance with an established protocolan assessment of medical records, laboratory results, patient history and death certificate information to determine the most plausible cause of death.The evaluation was based on individual case decisions in descending order, dependent from the level of consent with8.Using the above-mentioned diagnostic tools, the cases were evaluated and classified into categories adapted to Edler et al.30 after being fixed in toto in 4% formaldehyde, as well.Until autopsy, pmCT or usMIA the bodies were stored at 4 °C after death constantly. In case of short PMI (< 72 h) a standardized and extended tissue sampling was performed , an aerobronchogram and thickened pleural lines. In some cases, there were also subpleural indentations, as seen in peripheral pulmonary infarcts. Histological criteria of COVID-19 included diffuse alveolar damage (DAD), especially hyaline membranes, and activated pneumocytes, squamous metaplasia or organizing pneumonia.59. A high standard of diagnosis is ensured by many years of extensive experience in post-mortem and autopsy diagnosis. The diagnosis of a COVID-19 death required evidence of severe and fatal lung injury or other serious complications related to COVID-19 and excluded a competing cause of death, see Table At CA, a COVID-19 death was defined if the cause of death was found macroscopically within the pulmonary vasculature, in terms of embolism, or in the lungs as pneumonia and DAD. The cause of death was determined by postmortem examination, taking into account the medical history, if available, and any additional examinations in accordance with the guidelines of the German Society of Legal Medicine in its current versionNon-COVID-19 death were determined as acute and independently life-threatening conditions, such as pericardial tamponade.If no definite lung changes were found by any of the examination procedures or data was inconclusive, and no other manifest cause of death was apparent, the cases were classified as “unclear”.This was an exploratory hypotheses-generating study. Therefore, no confirmatory analyses were conducted. The p values were therefore not adjusted for multiplicity and are used exclusively as descriptive measures. The assumption of a normal distribution was checked graphically using range (IQR) and compared by the Mann–Whitney U test. Categorical variables are summarized as counts and percentages and compared by the chi-square test or Fisher’s exact test, as appropriate.Special attention was paid to the CA collective. In addition to above mentioned basic data, pre-existing medical conditions, body mass index (BMI) and autopsy findings were evaluated descriptively.Furthermore, a multiple logistic regression analysis was performed with COVID-19 death versus non-COVID-19 death as dependent variable, separately for the whole collective and the CA collective. The independent variables were age, sex and place of death, and for the CA collective, additionally, pre-existing medical conditions and BMI.11, as well as with the number of deaths in general in Hamburg in the past years (2000–2019) and per month (for the period 2016–2019)10.Finally, the COVID-19 deaths in Hamburg identified by the ILM were correlated with the total number of SARS-CoV-2 infections in Hamburg and the number of deaths presented by the RKIStatistical analysis and graphical presentation of the results were done using the statistical software SAS , GraphPad Prism® , SPSS®  and Microsoft Excel .Supplementary Information.


Vaccinium vitis-ideae and 1-year-old Calluna vulgaris under changing environmental conditions. The lowest radicle length and hypocotyl growth were observed in response to the effect of 1-year-old C. vulgaris and 2-year-old Rumex acetosella under increasing temperature (+4 °C) conditions. The chlorophyll a + b concentration in control seedlings strongly decreased from 0.76 to 0.66 mg g−1 . These factors may reduce the resistance of Scots pine to the effects of dominant species and affect the migration of Scots pine habitats to more favourable environmental conditions. Climate change influences the ecological environment and affects the recruitment of plants, in addition to population dynamics, including Scots pine regeneration processes. Therefore, the impact of cover-dominant species extracts on the germination of pine seeds and morpho-physiological traits of seedling under different environmental conditions was evaluated. Increasing temperature reinforces the plant-donor allelochemical effect, reduces Scots pine seed germination, and inhibits seedling morpho-physiological parameters. Conditions unfavourable for the seed germination rate were observed in response to the effect of aqueous extracts of 2-year-old  Climate change, in combination with other factors, influences the ecological environment; subsequently, this clearly affects the recruitment and population dynamics of plants . The earPinus sylvestris L.) has the largest geographical distribution among pine species, and it is one of the most widespread conifers on Earth, distributed from the Mediterranean to the Arctic [The capacity of plants to adapt to changing climatic and environmental conditions is becoming a pressing issue, and has practical importance. Adaptation capacity differs for various plant species . Climatee Arctic . In the e Arctic . Allelopathy, competition, soil nutrient imbalance, and poor ectomycorrhization have been implicated in conifer regeneration failure in the presence of dense ericaceous understory resulting from forest harvesting and fire in all ecosystem types. There are reports about allelochemical production in many woody species, such as in eucalyptus forests in Australia , in bore2 concentration changes, which are also associated with climate changes [2 concentration [2 and convert it into carbohydrates results in better plant growth and yield [2 fertilization effect and has received less attention compared with secondary climate change factors, such as increasing temperature [Within the forest floor litter, the dominant species of clear-cuts of Scots pine stands, which are mostly ericaceous, contain a range of secondary metabolites, mostly phenolic compounds, that are inhibitory to conifer seed germination, including primary root growth and ectomycorrhizal growth . This in changes ,23,24. I changes . Mittler changes , and Ain changes  reportedntration . The capnd yield . This isperature . TherefoVaccinio-Pinetum) stands in response to different temperature regimes and CO2 concentrations ex situ (in growing chambers). A complex experiment was designed to reveal the implications and interactions between allelopathic and plant developmental parameters (species and plant parts) in the germination of Scots pine seeds and morpho-physiological traits of seedlings.Laboratory studies of allelopathic effect are affected by the knowledge gap associated with imitating natural conditions to evaluate the same processes of allelopathic phenomena. However, research on this topic is widely performed by creating different conditions and setting different goals to better understand certain aspects of this plant-plant interaction. It is important to recognize that it is sometimes impossible to mimic climate changes during laboratory experiments, as the studied plants are sensitive to indoor cultivation. According to our previous studies and literature analyses, the aqueous extracts of dominant forest-floor species have an allelopathic effect, which may have an impact on the morpho-physiological parameters of Scots pine initial growth. Therefore, this study aimed to evaluate the impacts of aqueous extracts produced from both shoots and roots of 1-year-old and 2-year-old dominant plants grown in clear-cuts of Scots pine (2—400 ppm) and increasing temperature with higher CO2 concentrations   on Scots pine seed germination showed that the aqueous extracts of plant donors exhibited a higher allelopathic effect under increasing temperature  .2 concentration   . The ratis-idaea . V. vitis-idaea (p < 0.05) and R. acetosella (p < 0.05), and 1-year-old C. vulgaris (p < 0.05) shoot extracts when the temperature increased from 21.5/16 °C to 25.5/20 °C and the CO2 concentration increased from 400 ppm to 700 ppm  extracts and 2-year-old V. vitis-idaea (p < 0.05) shoot extracts on Scots pine seed germination rates when the temperature increased from 21.5/16 °C to 25.5/20 °C. The statistically significant lowest seed germination rate was recorded in the effect of the shoot extracts of C. epigejos (p < 0.05) under increasing temperature (25.5/20 °C) and higher (700 ppm) CO2 concentration conditions . The strongest inhibitory effect on the growth of Scots pine seedling radicles was determined in shoot aqueous extracts of 1-year-old V. vitis-idaea and P. schreberi, and of 2-year-old R. acetosella and C. epigejos. The results of our research (C. vulgaris (p < 0.05) and R. acetosella (p < 0.05) under increasing temperatures from 21.5/16 °C to 25.5/20 °C.According to RDA  analysisresearch  indicate2 concentration conditions , plant-donor aqueous extracts exhibited lower inhibitory effects on the hypocotyl length of Scots pine  and R. acetosella (p < 0.05). The radicle length of Scots pine seedlings significantly decreased due to the effect of the shoot aqueous extracts of C. epigejos (p < 0.05), R. acetosella (p < 0.05), 1-year-old V. vitis-idaea (p < 0.05), and P. schreberi (p < 0.05) under conditions of increasing temperature with higher CO2 concentration conditions   .R. acetosella and 1-year-old C. vulgaris under conditions of an increase in temperature from 21.5/16 °C to 25.5/20 °C , and an increase in temperature with higher (700 ppm) CO2 concentration  (The hypocotyl length of Scots pine seedlings significantly decreased due to the effect of shoot aqueous extracts of 2-year-old ctively) .a + b of Scots pine seedlings was significantly affected by the shoot aqueous extracts of P. schreberi (p < 0.05), V. vitis-idaea (p < 0.05), and C. vulgaris (p < 0.01) under conditions of an increase in temperature from 21.5/16 °C to 25.5/20 °C  and root aqueous extracts of C. vulgaris (p < 0.01) under conditions of increasing temperature with higher CO2 concentration . In addition, the increase in temperature significantly reduced the carotenoid content as a result of shoot aqueous extracts of 1-year-old P. schreberi (p < 0.01) and V. vitis-idaea (p < 0.01) and root aqueous extracts of 1-year-old C. vulgaris (p < 0.01) and 2-year-old C. epigejos (p < 0.01) (Plant-donor root aqueous extracts exhibited a lower negative impact on the carotenoid content of Scots pine seedlings . The hig < 0.01) .a + b content in Scots pine seedlings was observed in the effect of shoot aqueous extracts of P. schreberi (p < 0.01) under conditions of increasing temperature from 21.5/16 °C to 25.5/20 °C with higher CO2 concentration (700 ppm)  .a + b content of Scots pine seedlings due to the effect of root aqueous extracts of 1-year-old C. vulgaris (p < 0.01) and 2-year-old V. vitis-idaea (p < 0.01). The chlorophyll a + b content of Scots pine seedlings was more inhibited under conditions of increasing temperature with higher CO2 concentration  and root aqueous extracts of 2-year-old C. epigejos (p < 0.001) in increasing temperature with higher CO2 concentration conditions (C. epigejos (p < 0.01) and 1-year-old P. schreberi (p < 0.01) significantly changed the carotenoid concentration in Scots pine seedlings. This included root aqueous extracts of 1-year-old C. vulgaris (p < 0.01) and 2-year-old V. vitis-idaea (p < 0.01) under conditions of increasing temperature with higher CO2 concentration   .Variance analysis showed  that notp < 0.01) and hypocotyls (p < 0.05) were mostly affected by the interaction between the dominant species and environmental conditions in addition to the pH of the species extracts. The environmental conditions and the dominant species were the primary indicators that most significantly contributed to variability in the obtained estimates. In addition, it was determined that the pH of the species extracts significantly affected the germination rate (p < 0.01) and the carotenoid content (p < 0.05). A statistically significant low-positive correlation (r = 0.21) was determined between the extract pH and the germination rate. A low positive correlation (r = 0.25) was found between the extract pH and the carotenoid content.The radicle length  in high elevation forests have been reported to cause growth stagnation of conifers such as Tsuga plicata Donn, Thuja heterophylla (Raf. Sarge), Picea sitchensis, and Abies amabilis Dougl. [Vaccinium myrtillus L. has been reported to cause the regeneration failure of Picea abies (L) Karst. [The failure of natural regeneration and growth inhibition of planted conifers, such as black spruce, in the presence of  authors ,37. The s Dougl. ,39,40. I) Karst. . Phenoli) Karst. ,43. Expe) Karst. ,45. 2 concentrations [a/b-binding proteins, which could reduce the risk of photooxidative damage due to the relative decrease in the absorption cross-section of photosystems [2 concentration). Existing local adaptations that allow individuals to tolerate environmental conditions are especially important to increase the resistance and resilience of current populations. As mentioned previously, the chlorophyll content and the radicle length may be used as key parameters in studies because of their sensitivity to allelopathic compounds [The inhibitory effect of different plant-donor part extracts on photosynthetic pigment under different environmental conditions might be due to the imbalance in metabolism regulated by various enzyme activities . The strtrations . This prosystems . Alexievosystems  noted ouompounds . Plant aompounds .Any alterations in the plant community structure that are caused by climate change result from underlying changes in the population dynamics of the species that make up the community . In an ePinetum vacciniosum forests in June-July of 2015. In total, seventeen clear-cuts were evaluated, including eight one-year-old and nine two-year-old clear cuts in the Trakai Forest Enterprise in the south-eastern part of Lithuania . Aiming to determine the dominant species, the frequency (%) and relative plant cover (%) of ground vegetation species were estimated in 1 m2 subplots (n = 25) along transect in every 5 m in each site. The assessment was undertaken using the Braun–Blanquet [Arenosol [Pinetum vacciniosum, and the forest site type was Nb , according to the Lithuanian classification [The study of the successional stages of herbaceous plant and shrub phytocoenoses was conducted in one-year-old and two-year-old clear-cuts after reforestation of Arenosol . The forfication . The meaPleurozium schreberi and Vaccinium vitis-idaea . Calluna vulgaris was the third most dominant species in the 1-year-old clear-cut at a frequency of 87%. The relative plant cover of Vaccinium vitis-idaea increased by 2%,reached 12%, and maintained its dominant position in relation to other plants in the two-year-old clear-cuts. The highest frequencies in the 2-year-old clear-cut after reforestation were recorded for Calamagrotis epigejos and Rumex acetosella, of 44% and 68% respectively. These two species were classified as dominant due to their vigorous and various spreading abilities (both generative and vegetative), and these species structurally altered the phytocoenosis that was previously established in the clear-cuts.During the assessment of dominant species of the experimental plots in the one-year-old clear-cut after reforestation, the highest frequency was found to be of Pinus sylvestris L.) were assessed as the main indicators of the morphophysiological traits of dominants on the initial stages of forest regeneration in the clear-cuts.Subsequently, the dominant species (plant-donors) were used for production of aqueous extracts for the assessment of their allelochemical impact on potential acceptor species. Seed germination and seedling growth of the acceptor plant  were chopped into 2 cm sized pieces. Twenty grams of biomass were placed in glass bottles filled with 100 mL of distilled water; then, the samples were covered with foil, shaken several times, maintained for approximately 12 h at 18 to 20 °C, and filtered. This procedure was in a previous study .Pinetum vacciniosum forest type and soil conditions are similar to the current experiment’s soils. The composition of genotypes in seed orchards is broad in origin, so genetic diversity is also expected to be large. Pine seeds for the experiment were taken from the seed lots of several previous years and a mixture of seed orchards. The applicability of the results is defined by the forest type and soil conditions that actually prevail in pine stands in Lithuania. Pine stands occupy the largest forest area in Lithuania. Scots pine seeds were placed on filter paper in a Petri dish with a 9-cm-diameter. Five milliliters of 0.2% extract were added to each Petri dish and covered. For the control, 100 seeds were sown in distilled water. An additional 5 milliliters of distilled water and aqueous extracts from the dominant plants were added to Petri dishes every five days during the germination period. Each variant was performed in triplicate. The total number of Petri dishes was 324 .For the assessment, 100 sterilized Scots pine seeds were obtained from the Forest Seed and Plant Quality Department of the Lithuanian State Forest Service. It should be noted that Scots pine plus trees in Lithuania were selected mainly from the 2 concentrations on the allelopathic activity of dominant plants, the influence of different dominant species and plant parts on the germination and seedling growth of the Scots pine was assessed. The laboratory trials were carried out in the laboratory of Vytautas Magnus University. For the seed germination process, all Petri dishes were stored in chambers with controlled temperatures and CO2 conditions that consisted of three different temperatures and CO2 concentrations for 21 days. The photoperiod was 14 h, the air humidity was 75%, and the photosynthetic photon flux density was approximately 200 μmol m2 s−1 in all chambers. Light was provided by the combination of six natural fluorescent lamps  and one high-pressure sodium lamp (Philips MASTER GreenPower CG T 600 W). The Petri dishes were exposed to the following temperatures and CO2 concentrations:2,the first three chambers —temperature of 21.5/16 °C at day/night with 400 ppm CO2,the second three chambers (increasing temperature (+4 °C) conditions)—25.5/20 °C with 400 ppm CO2 concentration conditions)—25.5/20 °C with 700 ppm CO2.the third three chambers . The photoperiod, air humidity, and photosynthetic photon flux density were automatically adjusted. The temperatures and COMeasurements of Scots pine seedlings were performed. The germinated seeds were counted (in units) at day 21. Germination rates  were calculated relative to germination in the control (C) according to the formula :G = (Gv At day 21 the radicle and hypocotyl lengths (mm) were measured and deducted as a percentage of control as germination rate.a and b (chl a and b) and carotenoid assays. For the determination of pigment content, the method of Wettstein [a and b (mg g-1) was estimated spectrophotometrically using the T80 UV-VIS spectrophotometer  at wavelengths of 441, 662 and 644 nm (D), respectively. For the calculation of pigment content, the following formulas were used:c is the pigment content (mg g−1), V is the extract volume (mL), and P is the fresh mass of seedling needles (g).In addition, 0.2 g of fresh pine seedling shoots was sampled for chlorophyll ettstein  was usedettstein . The conLaboratory analyses were performed at the Institute of Forestry of the Lithuanian Research Centre for Agriculture and Forestry (LAMMC).F). The results of the allelopathic effects were statistically evaluated using the statistical package STATISTICA from Stat Soft. SAS UNIVARIATE procedure was used to check if residuals followed normal distribution. Based on the results on residual distribution, the Student t-test (t) or Kolmogorov–Smirnov (KS) test was used in pairwise comparison to determine statistically significant differences  in germination rates and morphophysiological parameters under different temperature regimes . The Kruskal–Wallis test was used for a group of treatments. Redundancy analysis (RDA) was used to determine relationships between different environmental conditions and the effects of dominant species and their parts on Scots pine germination rate and morphometric parameters . The SAS MIXED procedure (REML method) was used to obtain variance components of random effects and significances of fixed effects. The effects of fixed parameters, that is, temperature and the extract pH group  were evaluated using the TEST3 method.The significance of the data was assessed based on Wilkin’s λ-test and Fisher’s theoretical criterion  but with a single random effect. Therefore, we arrived at six models associated with respective random effects. This part of the statistical analysis was performed using the SAS software (SAS 9.4) [The initial SAS MIXED procedure was performed according to the following model.SAS 9.4) .2). However, the morphometric parameters of the seedlings remained unchanged. Higher air temperature resulted in a stronger negative effect, which decreased pine germination and seedling growth. The CO2 concentration of 700 ppm induced better growth of pine seedlings but decreased the concentration of photosynthetic pigments. The main effect on the germination of Scots pine seeds and morpho-physiological parameters of seedlings was induced by different environmental conditions and interactions between plant-donor species and climatic conditions. The assessment of climate conditions revealed the inhibitive properties of dominant extracts on seed germination under changing environmental conditions ex situ (a temperature of 25.5 °C during the day and 20 °C at night and 700 ppm of COIt can be assumed that the new findings obtained regarding the changes in the allelopathic potential of dominant species in the clear-cuts of previous Scots pine stands in Lithuania will improve the understanding of the impact of allelopathy on reforestation and the further management of forest ecosystems under changing climate conditions.


Introduction: To date, a universal validated and specific tool for assessing coronavirus disease 2019 (COVID-19) stigma among healthcare workers is lacking. We adapted a SARS stigma scale that was developed using the Berger HIV scale for use as a COVID-19 stigma scale and evaluated its psychometric properties among Egyptian physicians. Methods: We administered the 17-item SARS stigma scale in an anonymous online questionnaire among 509 Egyptian physicians recruited via convenience sampling during a cross-sectional study in June 2020. Exploratory factor analysis was performed on half of the sample. Confirmatory factor analysis of the resulting model was done using structural equation modeling on the other half. Scale reliability was examined using Cronbach’s alpha for internal consistency. Convergent construct validity was assessed using regression models to examine the association between the adapted COVID-19 stigma scale and relevant factors. Results: Exploratory factor analysis yielded 16 items (E16-COVID19-S) that supported a three-factor structure: personalized stigma (8 items); concerns of disclosure and public attitudes (5 items); and negative experiences (3 items). Cronbach’s α was 0.909 for the total scale and 0.907, 0.663, and 0.789 for the three subscales. E16-COVID19-S was confirmed to have good model fit . E16-COVID19-S was independently associated with physicians’ younger age, lower qualification, working in an isolation hospital, and self-stigma, whether the scale was treated as categorical or continuous. Conclusions: E16-COVID19-S exhibited good internal consistency and construct validity among this sample of Egyptian physicians. These adequate psychometric properties make the E16-COVID19-S scale appropriate for use by researchers and practitioners. The coronavirus disease 2019 (COVID-19) pandemic has been associated with various forms of stigma and discrimination since its beginning, being first mislabeled as the ‘Chinese virus’ . BesidesBeing at the frontline of the pandemic response, HCWs have been particularly affected by the COVID-19 pandemic and its associated distress . ApproxiAs of 10 April 2021, Egypt had 208,082 COVID-19 cases and 12,323 deaths [Mitigating COVID-19 social stigma is a timely necessity for upholding the psychological wellbeing and minimizing unfortunate outcomes among this much needed taskforce. In this context, early identification of COVID-19 stigma victims in this highly prone HCW population is crucial to enable prompt implementation of targeted interventions. To this end, a universal, validated COVID-19 stigma-specific tool is required for use by practitioners and researchers. However, such a tool is lacking locally and globally. To address this gap, we adapted a Severe Acute Respiratory Syndrome (SARS) stigma scale  that wasStudy design, procedures, and participants have been previously described in detail . BrieflyAt the time of the study, there were no specific published instruments that measured COVID-19 stigma. However, we found one tool that was developed by Verma et al.  that meaWe adapted the SARS stigma scale to measure COVID-19 stigma among this sample of Egyptian physicians. Experts in public health, occupational medicine, and psychiatry assessed the face and content validity of the adapted COVID-19 stigma scale. The scale was administered in English because Egyptian physicians are familiar with the language and they study Medicine in English. The scale was piloted in 10 physicians to assess whether the questions and answer categories were clear and appropriate. Based on feedback from physicians in the pilot study, one modification was performed to rephrase the word “touching me” to “getting close to me”. This analysis did not include pilot data.We hypothesized that other aspects of COVID-19 stigma that were asked about in the questionnaire and were experienced by this sample of Egyptian physicians would be associated with their stigma score based on our previous analysis and previous literature ,12,13,14This study was approved by the Ethical Review Board of Faculty of Medicine, Ain Shams University (FMASUR22/2020). All participants provided informed consent. Participation was voluntary, data were collected anonymously using serial study IDs, and data confidentiality was ensured.2 test of sphericity. The number of factors was inspected visually using the scree plot and was evaluated by examining the Kaiser Criterion of eigenvalues ≥1 [Exploratory factor analysis (EFA) was performed on one half of the sample and confirmatory factor analysis (CFA) on the other half to ascertain the factorial structure of the adapted COVID-19 stigma scale in this population of Egyptian HCWs. The sample was divided randomly using the automatic function in SPSS version 25. EFA was performed on the first dataset (n = 258) using principal component analysis. First, we checked the suitability of the data for factor analysis by performing the Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy and Bartlett’s χalues ≥1  and the alues ≥1 . Items walues ≥1 . Reliabialues ≥1 . Principalues ≥1 . The ite2/df < 2.00) was also assessed [Then, CFA was performed on the second dataset n = 251) using structural equation modeling (IBM AMOS version 16) to examine the fit of the model resulting from the EFA. The maximum likelihood estimation method was applied. Covariance was included among each pair of factors in the model. Unstandardized and standardized estimates of regression weights as well as covariances and correlations between variables were obtained, and modification indices were examined. As the data were non-normally distributed, we performed Bollen–Stine bootstrapping to test the null hypothesis that the model is correct. We report indices of model fit : the Tuc1 using sp-value ≤ 0.05 was used as the threshold for statistical significance.We conducted bivariable and multivariable analyses to examine convergent construct validity between the new COVID-19 stigma scale that was obtained from EFA and CFA (dependent variable) and its associations with the measures that were hypothesized to be associated with COVID-19 stigma (independent variables: self-stigma and anticipated stigma). The dependent variable was converted into percentages, and was then further categorized into three categories using tertiles , as Charles et al. proposed, because universal cut-off points that could be used for calculating stigma scores are lacking . In the General characteristics of the study sample of 509 Egyptian physicians have been previously reported . ParticiEFA was performed on the first half of the dataset. First, principal component analysis was performed, including all 17 items of the SARS stigma scale, using a cut-off point of 0.4 for factor loadings. Data were suitable for EFA as KMO was greater than 0.5 (0.916) and Bartlett’s test of sphericity was significant (<0.001). By examining the scree plot, a three-factor solution was suggested. Three factors yielded eigenvalues >1.0. A varimax rotation type of factor analysis was found to be suitable as the factors were orthogonal. The EFA was repeated with varimax rotation. Two items of the scale cross-loaded on Factors 1 and 2; their highest factor loadings were on Factor 1: “Some people who know I am a HCW have grown more distant” and “Most people nowadays are uncomfortable around a HCW”. One item cross-loaded on Factors 1 and 3; its highest factor loading was on Factor 1: “I have stopped socializing with some people because of their reactions to my being a HCW”. Next, reliability tests were done for the items that loaded on each factor and for each of the three factors. The reliability of Factor 2 was initially low (α = 0.365); removing the item that loaded on this factor, “I never feel the need to hide the fact that I am a HCW”, improved the reliability of Factor 2 (α = 0.663). EFA and reliability tests were repeated on the remaining 16 items of the scale. Factor loadings of the items on each of the three factors are shown in The reliability of the total 16-item COVID-19 stigma scale was α = 0.916. Eight items loaded on Factor 1 (α = 0.907), five items loaded on Factor 2 (α = 0.663), and three items loaded on Factor 3 (α = 0.789). This model explained 60.5% of the variance in the sample. Conceptually, the eight items loading on Factor 1 were related to personalized stigma; therefore, Factor 1 was labeled “personalized stigma”. The five items loading on Factor 2 were related to concerns of disclosure and public attitudes; thus, Factor 2 was labeled “concerns of disclosure and public attitudes”. Finally, the three items loading on Factor 3 were related to experiences that the physicians viewed as negative; hence, Factor 3 was labeled “negative experiences” .2 (94) = 167.167, χ2/df = 1.778, CFI =0.964, TLI = 0.953, RMSEA = 0.0056 and p-value = 0.235. Thus, this model was confirmed to have good model fit. All 16 items demonstrated distinctive and salient loadings ranging from 0.540 to 0.885 . By categorizing the continuous E16-COVID19-S scale into low, moderate, and high stigma levels using tertiles, approximately one third  of physicians reported a severe level of stigma. The mean E16-COVID19-S scale score was significantly higher among physicians reporting a severe level of stigma than among those reporting a mild or moderate level of stigma .p < 0.001). Physicians working in COVID-19 isolation hospitals were more likely to report a severe level of COVID-19 stigma compared with those working in other hospitals . Physicians who experienced self-stigma (feelings of guilt for exposing physician’s family to COVID-19 infection) and anticipated stigma (the need to hide physician’s COVID-19 positive test result) reported significantly higher rates of severe COVID-19 stigma than those who did not report self-stigma   .p = 0.021) and higher qualification , and was positively associated with HCWs’ work in isolation hospitals  and physicians’ self-stigma . Anticipated stigma was not independently associated with the E16-COVID19-S score  with a three-factor structure: personalized stigma; concerns of disclosure and public attitudes; and negative experiences. CFA confirmed that the E16-COVID19-S scale had good model fit. Using this scale, around a third of the studied physicians were found to experience severe COVID-19 stigma levels.To date, limited quantitative research exists on the magnitude of COVID-19 stigma among HCWs. Few studies reported varying proportions of COVID-19 stigma (ranging from 28 to 70%) ,12,13. HBy contrast, the E16-COVID19-S scale has preserved the original wording of the SARS stigma scale and consequently the HIV stigma scale upon which it was based. Additionally, the E16-COVID19-S scale was administered in the original English language, which makes its utilization possible for a wider base of practitioners and researchers worldwide. The E16-COVID19-S scale largely resembles the scale it was adapted from, the SARS stigma scale . HoweverThe E16-COVID19-S scale demonstrated good convergent construct validity with factors that were hypothesized to be associated with COVID-19 stigma, whether the scale was represented in tertiles or as a continuous variable, suggesting that these results were robust. Younger physicians’ age, lower qualification, working in a COVID-19 isolation hospital, and self-stigma were also found to be associated with COVID19 stigma in previous studies ,12,13,14This study presents a leading attempt to validate a specific COVID-19 stigma scale in HCWs. The sample consisted of physicians of various ages, work experience, and involvement in COVID-19 care pathways from different healthcare facilities in Egypt. The resultant E16-COVID19-S scale demonstrated acceptable psychometric properties: good model fit, high reliability, and convergent construct validity. The scale was administered in the English language, rendering it applicable for use in other settings, and was adapted from previous specific and validated stigma scales. These characteristics make the E16-COVID19-S appropriate for use in the early identification of stigmatized HCWs. Hence, it enables the implementation of prompt, targeted interventions to mitigate social stigma against HCWs.However, the E16-COVID19-S scale needs further validation across different HCW populations to examine its robustness and whether there is a need to modify its items/constructs. More in-depth investigations, parallel qualitative assessments, random rather than non-random sampling methods, and larger samples including various HCW categories would help to confirm and generalize these findings. The cross-sectional study design did not enable us to investigate the long-term reliability  of this scale. Future studies in this field using a longitudinal research design with at least two measurement points could assess this aspect. This sample consisted of physicians only. It is yet to be studied whether this tool is suitable for use in other HCW occupations and whether it would therefore need translation to local languages to address them more adequately. Nonetheless, physicians were found to be more prone to COVID-19-related stigmatization than other HCW occupations in other studies . PhysiciThe E16-COVID19-S is the first validated, specific COVID-19 stigma scale among HCWs. Validation of a specific COVID-19 stigma scale is a demand in the current global pandemic. Amid the third wave of this COVID-19 pandemic, the parallel increase in morbidity and mortality among HCWs along with the soaring numbers of cases has led to higher exposure to stigma and discrimination. The E16-COVID19-S scale can help in identifying HCWs facing COVID-19 stigma; thus, it will assist in supporting HCWs among their communities and protecting their mental health and social wellbeing.EFA in this population of Egyptian HCWs suggested a 16-item scale (E16-COVID19-S) with a three-factor structure: personalized stigma; concerns of disclosure and public attitudes; and negative experiences. CFA confirmed that the E16-COVID19-S scale had good model fit. The E16-COVID19-S scale was similar to the SARS stigma scale in its personalized stigma and disclosure concerns and concerns with public attitude domains. Almost one third of participating physicians experienced severe COVID-19 stigma. The E16-COVID19-S scale displayed good convergent construct validity with factors relevant to COVID-19 stigma such as younger physicians’ age, lower qualification, working in a COVID-19 isolation hospital, and self-stigma. The E16-COVID19-S scale demonstrated adequate reliability and validity and may assist practitioners and researchers in the early identification of HCWs who experience COVID-19 stigma to promptly facilitate their linkage to targeted interventions and services.